CN111518702A

CN111518702A - Helminthosporium umbilicalis strain, screening and identifying method and application thereof

Info

Publication number: CN111518702A
Application number: CN202010303803.XA
Authority: CN
Inventors: 强胜; 谭敏
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2020-08-11
Anticipated expiration: 2040-04-17
Also published as: JP2023522007A; US20230203429A1; CN111518702B; WO2021208989A1

Abstract

The invention discloses a Helminthosporium umbiliciforme strain, a screening and identifying method and application thereof. The Helminthosporium umbiliciforme strain (HXDC-1-2) is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 17778. The concentration of conidia using the strain was 10 per ml²More than one of the above plants can be used for biological weeding in rice, semen Maydis, semen glycines, semen Arachidis Hypogaeae, semen Sesami, semen Setariae, semen Viciae Fabae, semen Pisi Sativi, and oilThe herbicide composition is safe to use in fields of vegetables, flowers, traditional Chinese medicinal materials and fruit trees, is used for controlling gramineous weeds such as barnyard grass, moleplant seeds, large crabgrass, green bristlegrass, goosegrass herb, nit and seed grass, wild oats, false sorghum, soft-branch taraxacum, annual sedge and part of broadleaf weeds, and has a control effect of over 80 percent. As a biological herbicide, the weeding composition has the advantages of good weeding effect, low use amount, high safety to crops, low cost, environmental protection, no residue and no pollution.

Description

Helminthosporium umbilicalis strain, screening and identifying method and application thereof

Technical Field

The invention belongs to the technical field of application of microorganisms to agricultural plant protection and crop weed control, and particularly relates to a Helminthosporium umbiliciformis strain Bipolaris yamadae (Y.Nisik.) Shoemaker (HXDC-1-2), a screening and identifying method thereof and application thereof.

Background

Weed damage is one of the most significant factors contributing to crop losses, resulting in a loss of $ 950 million in agriculture worldwide each year. The area of Chinese farmland weed damage is about 7880 million hectares, and under the condition that 235 million yuan RMB weed control cost is invested every year, the loss of grain, cotton and oil is 1460 million, and the direct economic loss is nearly billion yuan RMB. Barnyard grass, wild oat, alopecurus, crab grass, moleplant seed, eleusine indica, green bristlegrass herb, chenopodium album and monochoria vaginalis are ten major weeds in farmlands, wherein 80 percent of gramineae. Since the beginning of agricultural production activities, attempts to control them have never been stopped, and in the early 50 s of the 20 th century, chemical herbicides have been rapidly developed due to their characteristics of convenient use, rapid onset of action, and the like. With the wide use of chemical herbicides, the negative effects are increasingly obvious. The environmental pollution crisis is brought by applying a large amount of chemical herbicide; the application of long residual effect herbicide causes residual toxicity and phytotoxicity, which leads to the yield reduction of the next crop and even the land degradation. Over 100 chemical herbicides have been banned or deregistered in over 30 countries around the world. The generation of drug-resistant weed populations, 324 biotypes of 188 weeds have been found to be resistant to 19 classes of chemical herbicides globally, resulting in reduced efficacy, increased dosage, increased cost, and increased pollution. The consequence is the simplification of herbicide varieties, the risk of resistance evolution is increased, and the technical requirements of the market on the research of novel broad-spectrum, high-efficiency and low-toxicity herbicides and the development of biological weeding are enhanced. The development of green herbicides, especially biological herbicides, as an alternative to chemical herbicides is an important approach to solve this conflict. In addition, the cost of developing a biological herbicide is tens or even hundreds of times lower than the cost of developing each new chemical herbicide up to 1 billion dollars. The development of this technology is driven by agricultural sustainability strategies implemented in many countries of the world, and the development of bioherbicides is a new approach to combating these grassy weeds. However, few effective bioherbicides are currently available to target these grasses.

The biological herbicide is a large dose of biological preparation applied to artificial culture and propagation under artificial control to kill weeds. Has two remarkable characteristics: firstly, a large amount of biological inoculants are obtained through manual mass production; the second is submerged application to achieve rapid infection and kill weeds in a short time. In 1981, DeVine was registered as the first bioherbicide in the United states. DeVine is chlamydospore suspending agent of Phytophthora palmae (Phytophthora palmivora) pathogenic strain growing in the state of Florida in the United states, is used for preventing and killing the weed Morrenia odorata (Morrenia odorata), has the prevention effect of 90 percent and the lasting period of 2 years, and is widely used in the state of orange gardens. Subsequently, Collego gets registered and put to practical use. The intervention of gene engineering and cell fusion technology can recombine excellent weed killing gene in nature, such as strong pathogenicity and toxin production, and provide people with the possibility of improving biological weed killing agent variety, raising control effect and improving host specificity. In addition to the 80 s development, Camperico and Biochon, launched at the end of the 90 s, have played an important role, even the only choice, in controlling the corresponding target weeds. The candidate biocontrol fungi which have been studied are relatively concentrated in the following genera: there are 18 species of anthrax (Colletotrichum), 13 species of Fusarium (Fusarium), 12 species of Alternaria (Alternaria), 8 species of Cercospora (Cercospora), Puccinia (Puccinia), Sclerotinia (sclerotirotinia), aleuromyces (Entyloma), chitomonas (Ascochyta). Major species of anthrax (Colletotrichum species) (US3,849,104 and US3,999,973) that are currently successfully used and patented in crop production; fusarium (Fusarium species) (US4,419,120); alternaria (Alternaria species) (US4,390,360); the genus Ascochyta species (US4,915,724) and the genus Sclerotinia species (CAOZ,292,233); sclerotinium rolfsii (SC64), and the like. At present, there are patents on the bipolaris setariae, the bipolaris maize and the bipolaris paniculata for biological herbicides in the bipolaris, there is no report on the bipolaris mountainous field, and there is no report on the application of the bipolaris paniculata in crop fields for biological control of gramineous weeds, lawn weeds, Genzhi taraxacum and kindred species thereof.

Disclosure of Invention

The invention aims to screen a mountain field pangolin helminthosporium strain for biological weeding, high efficiency, environmental protection, low cost, no pollution, safety for crops and obvious weeding effect, aiming at weeds (grassy weeds such as barnyard grass, green bristlegrass, large crabgrass, goosegrass, moleplant seeds, nit and seed plants, wild oats, alopecurus, false sorghum, soft-branch taraxacum and the like, annual sedge such as cyperus rotundus, cyperus rotundus and the like, and broadleaf weeds such as humulus scandens, medullcup, chenopodium album and the like) in crop fields such as rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetables, flowers, Chinese medicinal plants, fruit trees and the like.

In order to achieve the above purposes, the invention discloses a Helminthosporium umbiliciforme strain, a screening and identifying method and application thereof, and the adopted technical scheme is as follows:

the strain is Himalayan flatsedge helminthosporium HXDC-1-2, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 17778.

Preferably, the diameter of a bacterial colony of the bacterial strain is 50-60 mm, the bacterial colony is gray black, the back of the bacterial colony is black brown, and no water-soluble pigment exists; conidiophores of the conidiophores are olive green or yellow brown, are single or few and clustered, have light top color, are bent and bent like knees and have the width of 4.5-9.5 mu m; the conidiophores are yellow brown to dark brown, pseudo-spindle or inverted stick-shaped, straight or slightly curved, slightly wide in the middle, slightly narrow at the two ends, blunt and round in basal cells, smooth, 6-9 false septa, 54.5-92.5 multiplied by 12.5-17.5 microns.

A screening and identification method of Helminthosporium umbiliciforme strains comprises the following steps:

step 1) culturing the strain in a dark incubator at 28 ℃ for four days, extracting DNA by adopting a fungus gDNA extraction kit, and respectively carrying out PCR amplification by utilizing rDNA ITS sequences, GPDH sequences and EF1 alpha sequences, wherein universal primers are ITS4, ITS5, GPD1, GPD2, EF1-983F, EF 1-2218R;

ITS4：5’-TCCTCCGCTTATTGATATGC-3’

ITS5：5’-GGAAGTAAAAGTCGTAACAAGG-3’

GPD1：5’-CAACGGCTTCGGTCGCATTG-3’

GPD2：5’-GCCAAGCAGTTGGTTGTGC-3’

EF1-983F：5’-GCYCCYGGHCAYCGTGAYTTYAT-3’

EF1-2218R：5’-ATGACACCRACRGCRACRGTYTG-3’

step 2) carrying out agarose gel electrophoresis recovery on the amplification product obtained in the step 1), connecting the amplification product to a pMD19 vector, transforming Escherichia coli DH5 alpha, screening through blue and white spots, selecting white colonies, and shaking the white colonies to obtain a bacterial liquid;

step 3) sequencing the bacterial liquid prepared in the step 2), and performing homology comparison on ITS, GPDH and EF1 alpha related sequences of a nucleic acid database of a Genbank according to a measured result;

step 4) after amplification sequencing comparison, the homology of EF1 alpha amplification sequences MK026429 and KY905693 with the Bipolarisyamadae model strain ACCC36334 and CBS127087 respectively is more than 99 percent, the homology of ITS amplification sequence MF490813 with the Bipolaris yamadae model strain CPC28807 is more than 99 percent, the homology of GPDH amplification sequence MF490835 with the Bipolaris yamadae model strain CPC28807 is 99 percent, and the strain can be determined to be Helminthosporium yamami.

The above molecular marker can be used as a primer, and it can be detected that the homology of the above molecular marker sequence with the Bipolaris yamadae model strain CPC28807 is 99%, and the strain can be identified as the above strain, which is within the scope of the present patent protection.

Preferably, the ITS amplification sequence MF490813 is as set forth in SEQ ID NO: 1, and the GPDH amplification sequence MF490835 is shown as SEQ ID NO: 2, the EF1 alpha amplification sequences MK026429 and KY905693 are shown as SEQ ID NO: 3, respectively. But are not limited to the above molecular markers.

Use of a strain of Helminthosporium umbiliciforme as a biological herbicide.

Use of a strain of Helminthosporium umbiliciforme as a biological herbicide: the concentration of conidia of the mountain field bipolaris strain is 10 per ml²More than one, and is inoculated on the target weeds in the crop field.

Preferably, the weeds comprise grassy weeds, cyperaceae weeds and broadleaf weeds, wherein the grassy weeds comprise barnyard grass, green bristlegrass, large crabgrass, goosegrass herb, climbing fern, nits, wild oats, alopecurus, sorghum halepense, taraxacum komarovii and related genera; the Cyperaceae weeds include Cyperus rotundus, and Cyperus rotundus; the broadleaf weeds comprise humulus scandens, common sprouts, quinoa and small quinoa. But are not limited to the above weeds.

Preferably, the Brachypoda bambusicola kindred plants include taraxacum bambusicola, taraxacum japonica, hispid arthraxon.

Preferably, the crops comprise rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetables, flowers, traditional Chinese medicinal materials and fruit trees. But are not limited to the above crops.

Preferably, the vegetables comprise green vegetables, Chinese cabbage, radish, towel gourd, eggplant, and hot pepper; the Chinese medicinal materials include Perillae herba, herba Schizonepetae, and HUOXUEDAN; the fruit trees comprise waxberries. But not limited to the above vegetables, Chinese medicinal materials and fruit tree crops.

The invention has the following advantages and positive effects:

1. the special microbial inoculum of the paradise helminthosporium leptochloa bambusicola for biological weeding can be safely selected between crops and grassy weeds. The herbicide is environment-friendly, can inhibit the development of drug resistance and drug resistance of weeds, is beneficial to the popularization of green food and organic agriculture, and has the advantages of low cost, no pollution and safety to crops. Among the 103 crops or economic plants of 30 families tested, 33 plants are sensitive to Helminthosporium scoparium, 15 plants are slightly sensitive to Helminthosporium scoparium, and 5 plants are insensitive, wherein most sensitive plants are concentrated in the gramineae, and the sensitive plants are less sensitive to other plants such as leguminosae, cruciferae, benaceae and part of gramineae. Therefore, the strain can be used for crop fields of rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetable, flower, traditional Chinese medicine, fruit trees and the like, and is used for controlling farmland weeds including gramineous weeds such as barnyard grass, green bristlegrass, large crabgrass, goosegrass, moleplant seed, nitand grass, wild oat, alopecurus, false sorghum, Gentiana minor and the like, annual sedge such as cyperus rotundus, cyperus rotundus and the like, and broadleaf weeds such as humulus scandens, Aeschynia indica, quinoa, chenopodium quinoa and the like.

2. The mountain field helminthosporium peregrinum strain is applied to biological weeding, utilizes living organisms which are directly collected in a natural environment and are natural pathogenic bacteria of target weeds generated in a target crop field and the environment, has no ecological risk in the use process due to the existence in the environment, has strong specificity, high safety to crops and other non-target plants, does not cause residual harm to the environment, is quickly decomposed and degraded after the thalli die, and can be used for producing green or organic agricultural products because degradation products are all recyclable organic matters and cannot cause pollution.

3. The control effect of the strain on target weeds can reach more than 80%, and the strain is particularly suitable for plants in seedling stage.

4. According to the test, the mountain field helminthosporium umbiliciformis HXDC-1-2 is safe for most crops, has moderate sensitivity only to sorghum, has weak sensitivity to cotton, wheat and barley, can be safely used in rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetable, flower, Chinese medicinal materials and fruit tree crop fields, and can be used for controlling weeds in the fields (including barnyard grass, green bristlegrass, digitaria sanguinalis, eleusine indica, moleplant seed, nitre, wild oat, alopecurus, sorghum halepense, taraxacum and other gramineous weeds, cyperus rotundus and other annual sedge weeds and also broad leaved weeds such as humulus scandens, medullus, chenopodium quinoa and small gooseberry). This again indicates that Helminthosporium umbiliciforme HXDC-1-2 has the potential to be developed into a biological herbicide for preventing and killing off Brachypoda bambusicola and gramineous weeds.

Drawings

FIG. 1 is a colony morphology of HXDC-1-2 Helminthosporium umbiliciforme strain;

FIG. 2 shows conidiomorphism of HXDC-1-2 Helminthosporium umbiliciforme strain;

FIG. 3 is a gel electrophoresis of HXDC-1-2 Helminthosporium umbiliciforme strain;

in FIG. 3, 1 and 2 are EF 1. alpha. sequences, 3 and 4 are ITS sequences, and 5 and 6 are GPDH sequences.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

A Helminthosporium umbilicalis strain Bipolaris yamadae (Y. Nisik) Shoemaker (HXDC-1-2) (collection unit: China general microbiological culture Collection center; address: Beijing's republic of the sunward area, North Chen West Lu No.1, Collection date: 5 and 10 days in 2019; collection No. CGMCC No.17778), Hippocampus strain belongs to the kingdom Fungi (Fungi), Ascomycota (Ascomycota), Ascomycetes (dothieomycetes), Geospora (Pleospora citridae), Geospora (Pleospora Barttrella), Geospora (Plesponheimeridae), Cochliomycetes (Cochliella Drech) [ stage: helminthosporium applanatum (Bipolaris shoehorn), helminthosporium alpinum (Bipolaris yamadae (y. nisik) Shoemaker). The growth is fast on a potato glucose culture medium, the culture is carried out for 5 days under the dark condition at the temperature of 28 ℃, the diameter of a bacterial colony is 50-60 mm, the bacterial colony is gray black, the back of the bacterial colony is dark brown, and no water-soluble pigment exists. FIG. 1 shows the colony morphology of Helminthosporium umbiliciforme after 5 days of culture. The conidiophores are green or yellow brown, single or few clusters, light in top color, bent and bent like knees and 4.5-9.5 mu m wide. Conidia are yellowish brown to dark brown, pseudo-spindle-shaped or inverted stick-shaped, straight or slightly curved, slightly wide in the middle, slightly narrow at both ends, blunt and round in basal cells, smooth, 6-9 (mostly 7-8) pseudo-septa, 54.5-92.5 × 12.5-17.5 μm (average 69.5 × 14.7 μm). FIG. 2 shows the conidiomorphism of Helminthosporium umbilicalis under high power microscope.

Example 1

The new strain is obtained by the following ways, and the inventor investigates the disease occurrence of the soft-branch atracty bamboo in places such as Anhui, Fujian, Guangdong, Guangxi, Guizhou, Henan, Hubei, Hunan, Jiangsu, Jiangxi, Sichuan, Shandong, Taiwan, Yunnan, Tibet and Zhejiang province, grassland, open field, wetland, crop field edge, ditch, hedgerow, canyon and farmland, collects the diseased soft-branch atracty bamboo leaf, records the disease symptoms and related information of plants, brings the plants back to a laboratory, and carries out separation research on parasitic fungi and carries out pathogenicity verification strictly according to Koch' spontulate.

Firstly, the pathogenic symptoms of spores on the Brachypodium amazonicum generated by the natural condition of the Shantian Helminthosporus strain (Bipolaris yamadae (Y.Nisik.) Shoemaker) HXDC-1-2 are as follows: the leaves of the diseased plants of the Gentiana minor can be seen with disease spots of different sizes, in the early stage of disease, dark brown or black disease spots appear on the surfaces of the plant leaves, circular (the diameter is 0.25-0.40 cm) or oval (the width is 0.25-0.3 cm multiplied by the length is 0.4-0.5 cm) disease spots, yellow or green halos exist on the edges, and the leaf tips and the leaf edges are yellowed. In the middle stage of the disease, the lesion is gradually enlarged, the edge of the diseased leaf is withered yellow to brown and slightly curly, and the center of part of the leaf is changed into yellow to brown. The disease spots are connected into a large brown or black patch, and the leaves are withered yellow or curled until the whole leaf plant dies.

Tissue blocks are cut at the junction of diseased leaves and surface sterilized with 75% alcohol and 1% sodium hypochlorite solution. Inoculating the small tissue blocks on PDA culture medium, culturing in dark at 28 deg.C for 2 days, and purifying the separated strains to obtain the final product with hypha growth and spore production similar to those of the previous one and strong pathogenic bacteria of Pleioblastus Amarus. It was stored on a slant medium and kept at 4 ℃ for further use.

The shapes of conidia and conidiophores are observed under an optical microscope, and meanwhile, the sizes of the conidia are measured by photomicrographs. According to the morphological characteristics described in Chinese journal of fungus and handbook of identifying fungus, the pathogeny is identified. The morphological characteristics of the strain HXDC-1-2 are identified as Bipolaris yamadae, which is basically consistent with the description of Helminthosporium umbiliciforme (Y. Nisik.) Shoemaker in volume thirty of Chinese journal of fungi. The description is as follows: conidiophores are yellow brown, are single or clustered, are sometimes branched, are light in top color, bent like knee and bent, and have a width of 4.5-9.5 mu m. Conidiophores are yellow brown to dark brown, pseudo-spindle or sometimes inverted stick-shaped, straight or slightly curved, slightly wide in the middle, blunt and round in basal cells, smooth, 6-9 (mostly 7-8) pseudosepta, 54.5-92.5 × 12.5-17.5 μm (average 69.5 × 14.7 μm); umbilicus is obvious and slightly prominent.

Extracting pathogenic fungi DNA by using a fungi gDNA extraction kit (BIOMIGA), and respectively performing PCR amplification by using an rDNA ITS sequence, a GPDH sequence and EF1 alpha. The universal primers are ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3'), GPD1 (5'-CAACGGCTTCGGTCGCATTG-3'), GPD2 (5'-GCCAAGCAGTTGGTTGTGC-3'), EF1-983F (5 '-GCYCCYGGHCAYCGTGAYTTYAT-3'), EF1-2218R (5 '-ATGACACCRACRGCRACRGTYTG-3'), respectively. And (3) recovering an amplification product through agarose gel electrophoresis, connecting the amplification product to a pMD19 vector, transforming escherichia coli DH5 alpha, screening through blue white spots, selecting white colonies, and shaking bacteria. Performing PCR amplification, detecting with electrophoresis and gel analyzer, and displaying a single bright band on 2% agarose gel, as shown in FIG. 3, wherein 1 and 2 in the figure are EF1 alpha amplification sequences, and the size of the target band is about 800 bp; 3. 4 is an ITS amplification sequence, and the size of a target band is about 500 bp; 5. and 6 is a GPDH amplification sequence, and the size of a target band is about 500 bp. Sequencing was performed by Shanghai Biotech, and homology comparison was performed on EF1 alpha, ITS and GPDH related sequences in Genbank nucleic acid database (Blast software analysis). After amplification sequencing alignment, the homology of the EF1 alpha amplification sequence (MK026429 and KY905693) and the Bipolaris yamadae model strain (ACCC36334 and CBS127087) is 99 percent respectively, the homology of the ITS amplification sequence (MF490813) and the Bipolaris yamadae model strain (CPC28807) is 99 percent, the homology of the GPDH amplification sequence (MF490835) and the Bipolaris yamadae model strain (CPC28807) is more than 99 percent, and the strain is determined to be Helicoverpa zeae. ITS amplification sequence (MF490813) is shown in seq id NO: 1, GPDH amplification sequence (MF490835) is shown as SEQ ID NO: 2, the EF1 alpha amplification sequence (MK026429, KY905693) is shown as SEQ ID NO: 3, respectively.

The pathogenic symptoms of spores on the soft branch taraxacum bambusicola, which are produced by pure culture of the mountain field helminthosporium HXDC-1-2, are as follows: obvious dark brown or black scabs appear on the surface of the blade, the blade is round or oval, part of the blade tip or the blade edge is yellowed, the scabs are gradually enlarged and the color is deepened along with the lapse of time, the scabs of the blade are dense and flaky when the disease is serious, the top end of the blade is withered, most of the blade is yellowed or withered at the late stage of inoculation, and the blade is curled. And (4) re-separating the pathogenic bacteria from the diseased leaves, wherein spores on the surfaces of diseased spots are consistent with the spores of the sprayed mountain field helminthosporium umbiliciformis, and the Cochh rule is met.

The onset symptoms under the above experimental conditions are similar to those of natural disorders.

The camptothecellus strain HXDC-1-2 is separated from the brachymystax lenok, but has broad-spectrum pathogenicity on gramineous weeds, and also has the advantage that a plurality of annual and perennial gramineous weeds can be effectively killed without influencing peripheral crops.

Characterized by being as follows: ascomycota (Ascomycota), Ascomycota (Dothidymycoetes), Geospora (Pleosporomycotidae), Geospora (Pleospororales Luttrell ex Barr), Geosporaceae (Plesporaceae Nitschke), Cochliobolus (Cochliobolus Drechsler) [ asexual stage: helminthosporium umbilicalis (Bipolaris shoehorn) ], helminthosporium umbilicalis (Bipolaris yamadae (y. nisik.) Shoemaker). The specialized strain mainly infects the taraxacum leptostachya and its kindred species and grassy weeds, is highly immune to main crops such as rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetable, flower, traditional Chinese medicinal material and fruit tree, has 33 species sensitive to the mountain field Helminthosporium amplexicaule, 15 species slightly sensitive to the mountain field Helminthosporium and 5 species insensitive to the mountain field Helminthosporium in the tested 30 families of 103 crops or economic plants, wherein the sensitive plants are mostly concentrated in grassy and have weaker sensitivity to other families of plants such as leguminous, cruciferae, sesamum and part of grassy plants. Therefore, the strain can be used for crop fields of rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetable, flower, traditional Chinese medicine, fruit trees and the like, and is used for controlling farmland weeds, including gramineous weeds such as barnyard grass, green bristlegrass, digitaria sanguinea, goosegrass, moleplant seed, nitand grass, wild oat, alopecurus, false sorghum, soft-twig taraxacum and the like, annual sedge such as cyperus rotundus, cyperus rotundus and the like, and broadleaf weeds such as humulus scandens, Aeschynia indica, quinoa, chenopodium quinoa and the like.

Example 2

The mountain field helminthosporium umbiliciformis HXDC-1-2 is used for biological weeding, and the specific application method comprises the following steps: inoculating the strain in the following culture medium, inducing to generate conidium, spraying in field or weed generating place, and using dosage is 10²～10⁶one/mL.

The culture method comprises the following steps: the method comprises the steps of growing the Helminthosporium umbiliciforme in the dark for about 4 days, placing a culture dish under the aseptic condition under a black light lamp or an ultraviolet lamp (24h) for irradiation, and enabling a culture medium to generate spores in the irradiation process.

PDA culture medium-potato glucose agar culture medium

At present, no report about the fungus herbicide of the Gentiana mollissima exists at home and abroad, and the research on the aspect provides a new excellent way for preventing and killing the Gentiana mollissima and the gramineous weeds. The weeding composition can be matched with other suitable chemical herbicides for use, so that the dosage of the chemical herbicides is reduced, and the pollution to the environment is reduced.

According to the needs, the herbicidal composition of the present invention can use surfactants, stabilizers and other adjuvants, surfactants such as tween 20, tween 80 and the like, and stabilizers such as antioxidants and the like.

Example 3

Collecting the leaves of diseased soft branches of taraxacum officinale from the field, recording the disease symptoms of the plants, and performing microscopic examination. Separating pathogenic fungi with Armillaria leptostachya soaking agar culture medium. Single spores of each strain are picked under a microscope, inoculated on a PDA culture medium respectively, cultured in the dark at 28 ℃, and the colony characteristics of pure culture of the pathogenic fungi are observed and the colony diameter is measured. Another colony disk with a diameter of 5mm was inoculated in the center of the PDA plate, and the diameter of each colony was measured after 4 days.

TABLE 1 comparison of colony characteristics of Pleioblastus gramineus

Note: d1 represents the diameter of the colony formed after 5 days of monospore,

d2 represents the diameter of colonies formed after 4 days on 5mm colony discs.

Example 4

Pathogenic fungi are collected and separated from the disease plants of the Rouzhi taraxacum which naturally occur in Guiyang of Guizhou, and the pathogenicity of the pathogenic fungi is tested. Spraying Gentiana Robusta with spore suspension of various pathogenic fungi (surfactant including Tween 20 and Tween 80, and stabilizer including antioxidant) at different concentrations under natural conditions, and observing pathogenic characteristics of each fungus.

TABLE 2 comparison of the virulence of several pathogenic fungi isolated from Gentiana mullerica plants on Gentiana mullerica

Note: 10⁵、10⁴The spore suspension concentrations for spraying are respectively expressed as 10 per ml⁵、10⁴Spores (same below).

The lower case letters were at a significance level of 0.05 and the upper case letters were at a significance level of 0.01 (the same below).

As can be seen from Table 2, the pathogenicity rate of the Helminthosporium umbiliciforme strain HXDC-1-2 is higher than that of other strains.

Example 5

The HXDC-1-2 strain was cultured on PDA medium for 4 days, irradiated with a black light at 28 ℃ and then suspended in sterile water (same as in example 4). And culturing the young seedlings of the Gentiana mollissima in a 9cm pot, spraying a spore suspension by using a handheld sprayer when the young seedlings grow to 3-4 leaves, keeping the young seedlings at 28 ℃ for 48 hours, keeping the moisture of the young seedlings at 80%, and completely killing the weeds after the young seedlings are treated for one week.

Example 6

Sowing seeds of test plants (shown in tables 3 and 4) in pots with the diameter of 9cm, setting 4 groups of repetition, culturing in a greenhouse at the temperature of 28 ℃, inoculating when the test plants reach the leaf stage of 3-4 (same as examples 4 and 5), reserving 5-10 plants in each pot, and testing the sensitivity of HXDC-1-2 strains to each test plant. The safety test result shows that the HXDC-1-2 strain has pathogenicity on sorghum and cowpea, and has no pathogenicity on rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetable, flower, traditional Chinese medicinal material, fruit tree and other crops. The results of the herbicide controlling spectrum test show that the HXDC-1-2 strain has strong pathogenicity on gramineous weeds such as cockspur grass, green bristlegrass, large crabgrass, goosegrass herb, moleplant seed, nitre, wild oat, alopecurus, sorghum halepense, taraxacum, cymarosa, cyperaceae weeds such as cyperus rotundus, broadleaf weeds such as humulus scandens, Aeschynia indica, Chenopodium album and Chenopodium quinoa, and has moderate pathogenicity on the large crabgrass, the goosegrass herb, the heteroclite and the like. Therefore, the HXDC-1-2 strain can be applied to crop fields of rice, corn, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetables, flowers, Chinese medicinal materials, fruit trees and the like, and can be used for preventing and treating gramineous weeds such as barnyard grass, green bristlegrass, digitaria sanguinea, eleusine indica, moleplant seed, nit grass, wild oat, alopecurus, sorghum halepense, soft-branch taraxacum and the like, annual cyperus rotundus such as cyperus rotundus, cyperus rotundus and the like, and broadleaf weeds such as humulus scandens, Aeschynia indica, quinoa, chenopodium quinoa and the.

Sequence listing

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ttaagttcag cgggtatccc tacctgatcc gaggtcaaaa gttaaaattt gtagagtctt 60

gatggattac cgtccttttc tcctgacaca gagtgcaaaa tatgtgctgc gctccgaaac 120

cagtaggccg gctgccaatc gttttaaggc gagtctccca gcaagaggga gacaaaaaac 180

gcccaacacc aagcaaagct tgaaggtaca aatgacgctc gaacaggcat gccctttgga 240

ataccaaagg gcgcaatgtg cgttcaaaga ttcgatgatt cactgaattc tgcaattcac 300

actacgtatc gcatttcgct gcgttcttca tcgatgccag aaccaagaga tccgttgttg 360

aaagttgtaa taattacatt gttttttact gacgctgatt gcaactgcat tagaaaaagg 420

tttatggttt ggtcctggtg gcgggcgaac ccgcccagga aacaacaagt gcgcaaaaga 480

catgggtgaa aaaaatattt cagccggccg cgaagccaaa gccttcatat tttcgttgtg 540

taatgatccc tccgcaggtt cacctacgga ga 572

<210>2

<211>577

<212>DNA

<213> Helianthus platyphylla (Bipolaris yamadae)

<400>2

aagaggcgtt ggagagtacc tcgatgtcgg gcttgtaggt ctcgtggttg acacccatga 60

cgaacatggg ggcgtcagcg gagggagcag agatgacaac cttcttggct ccgcccttca 120

agtgggcctt ggccttctcg gtggtggtga agacaccggt agactcgacg acgtagtagg 180

cgccagtctc gctccatgga atgttggcgg ggtccttctc catgtggaaa cggatagtct 240

tgccgttgac ggtcaggttg ttgccgtcaa ccttgatgtc acccttgaac tggccgtgtg 300

tgctgtcata cttgagcatg tatgcctgtg tatacgtcag tctgcatggt tccatcaaag 360

aaatgacacc agtgcgtcag gccgaagcag acgcttgctg tgatgaaagg ttctgggttg 420

agggagtgct tacagcgtag tggggctcga tgaaagggtc gtttacggcg acaatgtcga 480

cgtcgttgtg ctcgatgctg agatatgggg tcagctttgg tgtgcgtaaa atggacaaac 540

cccaaggata cttacgcatt gcggaagacg atgcggc 577

<210>3

<211>770

<212>DNA

<213> Helianthus platyphylla (Bipolaris yamadae)

<400>3

caagaacatg atcactggta cctcccaggc tgactgcgct atcctcatta tcgctgccgg 60

tactggtgag ttcgaggctg gtatctccaa ggatggccag actcgtgagc acgcccttct 120

cgcctacacc cttggtgtca agcagctcat cgttgccatc aacaagatgg acaccaccaa 180

gtggtctgag gaccgttacc aggagatcat caaggagacc tccaacttca tcaagaaggt 240

cggctacaac cccaagcacg ttcccttcgt gcccatctcc ggtttcaacg gtgacaacat 300

gattgaggcc tccaccaact gcccctggta caagggttgg gagaaggaga ccaagtccaa 360

ggccaccggt aagaccctcc tcgaggccat cgatgccatc gaccctccca gccgtcctac 420

cgacaagccc ctccgtcttc ccctccagga tgtgtacaag atcggtggta ttggcacggt 480

tcccgtcggt cgtgtcgaga ccggtatcat caaggccggt atggtcgtca ccttcgcccc 540

cgctggtgtc accactgaag tcaagtccgt cgagatgcac cacgagcagc tgaccgaggg 600

tgtccccggt gacaacgtcg gcttcaacgt caagaacgtc tccgtcaagg agatccgtcg 660

tggtaacgtt gctggtgact ccaagaacga cccccccaag gcttccgagt ccttcaacgc 720

ccaggtcatc gtcctcaacc accccggtca ggtcggtgcc ggttacgcac 770

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

tcctccgctt attgatatgc 20

<210>5

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ggaagtaaaa gtcgtaacaa gg 22

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

caacggcttc ggtcgcattg 20

<210>7

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gccaagcagt tggttgtgc 19

<210>8

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gcyccygghc aycgtgayttyat 23

<210>9

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atgacaccra crgcracrgt ytg 23

Claims

1. The mountain field helminthosporium umbiliciformis strain is HXDC-1-2, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation number of CGMCC No. 17778.

2. The Helminthosporium umbiliciforme strain of claim 1, wherein the diameter of the colony of the strain is 50-60 mm, the colony is dark black, the back of the colony is dark brown, and no water-soluble pigment exists; conidiophores of the conidiophores are olive green or yellow brown, are single or few and clustered, have light top color, are bent and bent like knees and have the width of 4.5-9.5 mu m; the conidiophores are yellow brown to dark brown, pseudo-spindle or inverted stick-shaped, straight or slightly curved, slightly wide in the middle, slightly narrow at the two ends, blunt and round in basal cells, smooth, 6-9 false septa, 54.5-92.5 multiplied by 12.5-17.5 microns.

3. The method for screening and identifying Helminthosporium umbiliciforme strain according to claim 1, comprising the steps of:

ITS4：5’-TCCTCCGCTTATTGATATGC-3’

ITS5：5’-GGAAGTAAAAGTCGTAACAAGG-3’

GPD1：5’-CAACGGCTTCGGTCGCATTG-3’

GPD2：5’-GCCAAGCAGTTGGTTGTGC-3’

EF1-983F：5’-GCYCCYGGHCAYCGTGAYTTYAT-3’

EF1-2218R：5’-ATGACACCRACRGCRACRGTYTG-3’

step 2) carrying out agarose gel electrophoresis recovery on the amplification product obtained in the step 1), connecting the amplification product to a pMD19 vector, transforming Escherichia coli DH5 alpha, screening through blue and white spots, selecting white colonies, and shaking the white colonies to obtain a bacterial liquid; step 3) sequencing the bacterial liquid prepared in the step 2), and performing homology comparison on ITS, GPDH and EF1 alpha related sequences of a nucleic acid database of a Genbank according to a measured result;

4. The method for screening and identifying Helminthosporium umbiliciforme strain according to claim 3, wherein the ITS amplification sequence MF490813 is as shown in SEQ ID NO: 1, and the GPDH amplification sequence MF490835 is shown as SEQ ID NO: 2, the EF1 alpha amplification sequences MK026429 and KY905693 are shown as SEQ ID NO: 3, respectively.

5. Use of the Helminthosporium umbiliciforme strain of claim 1 as a biological herbicide.

6. Use of the Helminthosporium umbiliciforme strain of claim 5 as a biological herbicide, wherein the concentration of conidia of the Helminthosporium umbiliciforme strain is 10 per ml²More than one, and is inoculated on the target weeds in the crop field.

7. Use of the mountain field helminthosporium applanatum strain as a biological herbicide according to claim 6, characterized in that the weeds comprise grassy weeds, cyperaceae weeds, broadleaf weeds, wherein the grassy weeds comprise barnyard grass, green bristlegrass, large crabgrass, goosegrass, moleplant seeds, nits and seeds, wild oats, alopecurus, sorghum halepense, taraxacum malabaricum and allium plants thereof; the Cyperaceae weeds include Cyperus rotundus, and Cyperus rotundus; the broadleaf weeds comprise humulus scandens, common sprouts, quinoa and small quinoa.

8. Use of the strain of Helminthosporium umbilicifolius according to claim 7 as a biological herbicide, wherein the Brachypoda bambusicola kindred plants include taraxacum atractylis, taraxacum bambusicola, Arthropoda japonica, Hispanica.

9. Use of the mountain field helminthosporium umbilicifolium strain as a bioherbicide according to claim 6, characterized in that the crops comprise rice, maize, soybean, peanut, sesame, millet, broad bean, pea, rape, vegetables, flowers, chinese medicinal materials, fruit trees.

10. Use of the Helminthosporium umbiliciforme strain of claim 9 as a bioherbicide, wherein the vegetables comprise green vegetables, chinese cabbage, radish, luffa, eggplant, capsicum; the Chinese medicinal materials include Perillae herba, herba Schizonepetae, and HUOXUEDAN; the fruit trees comprise waxberries.